Science in Action: Part I. Engineering Plants Using Crown Gall Disease

Ever since Mendel discovered that
characteristics in pea plants could be inherited, scientists have been
improving plants through hybridization; two related plants were crossed and the
resulting offspring had characteristics of both parent plants. Breeders then
selected and reproduced the offspring that had the desired traits. These
conventional plant breeding techniques were relatively imprecise because they
shuffled thousands of genes around and distributed them to the offspring just
to get one important change in a plant that was economically worth pursuing.

One
challenge encountered in Mendelian breeding is that generally only closely
related species of plants could be crossed. If no closely related species with
desirable traits existed, breeders had no way of passing on these traits to the
other plant. Another problem was that some of the genes were linked to each
other. This is seen today in tomatoes that have been bred so that they can be
shipped long distances but with a substantial loss in flavor.

Since the early 1980’s scientists
have been using the tools of modern biotechnology to insert a single gene, or
just two or three genes, into a plant giving it new, advantageous
characteristics. With this technology a single gene could be inserted into a
plant giving it a desired characteristic instead of the mixing all the genes
from two plants through traditional plant breeding and hoping for the best.
This technique could develop a new plant with much more control and precision
and at a rate much faster than ever before.

The
bioengineering of plants emerged from discoveries by researchers in previous
years on how bacteria caused plant tumors, how viruses protected plants from
other viruses and what enabled some bacteria to kill insects. Some
first major step toward biotechnology occurred early in the twentieth century
with a plant disease called crown gall. Crown galls are tumor-like plant
growths that occur on many woody plants including fruit trees, grape vines, and
ornamentals. In 1907 researchers at the USDA discovered that the cause of crown
galls was a soil bacterium,
Agrobacterium
tumefaciens. Other bacteria were known to
cause plant diseases but A. tumefaciens had the unusual ability to cause
the plant that was hosting it to grow a disfiguring tumor.

Forty years later in 1947
researchers at the Rockefeller Institute for Medical Research (now named Rockefeller
University), curious about the crown gall bacterium and using it for
insight into how tumors developed, grew crown gall tissue culturally free of
any associated bacterium AND free of the plant host as well. They found that
these uninfected crown galls could continue to grow, as crown gall tissue,
independently of the crown gall bacterium and of the plant host for many years.
It was concluded that normal plant growth in some unknown way had been
permanently and irretrievably transformed by A. tumefaciens.
Understanding how would have to wait nearly another thirty years.

During the 1950s and 1960s, scientists
discovered DNA’s
role in transmitting information from plant to plant and ultimately controlling
plant growth. Armed with this new information, scientists began looking more
closely at DNA’s role in the formation of crown gall. The crown gall mystery
was attacked again when several investigators began, logically, looking for the
tumor-inducing factor in the bacterium’s DNA.

Bacterial DNA
is relatively simple compared to other types of organisms since it can be
normally found on a single chromosome.
It wasn’t found there. Instead it was found by Flemish researchers on a
smaller, mobile DNA unit called a plasmid that was not part of the bacterium’s
single chromosome. In a series of experiments at the University of Washington
ending in 1977 researchers found that this bacterial plasmid was spliced into
the chromosomes of plant cells when the bacteria infected the plants.

This was at the same time that
researchers in other fields were just beginning to understand how to manipulate
genetic information by a technique called, in lay terms, gene splicing – to cut
and splice foreign DNA into the genetic code of an organism.

It became clear now that A.
tumefaciens transmitted the genetic information needed to cause a plant to
produce tumor-like growth through the transfer of a “packet” of information
called a plasmid.  This plasmid altered
the genetic makeup of the plant so that the infected cells of the plant were
induced to divide continually, developing galls containing the genetic
information from A. tumefaciens.

What if scientists could manipulate
A. tumefaciens so that it no longer
transferred the genetic information for creating crown gall but instead
transferred genetic information into plants that produced desirable traits such
as resistance to insects or disease?

To convert the A. tumefaciens
plasmid into a beneficial plasmid (now called a vector) researchers first
had to locate and then replace the tumor-inducing genes. By 1983, plant
molecular biologists had developed the first plasmid vector for plants
susceptible to crown gall from A.
tumefaciens. The crown gall disease had changed plant breeding forever.

A
tool for introducing genes
into plants is useful only if scientists have found genes that they want to
transfer. Enter Monsanto. In the late 1960’s researchers at Monsanto wanted to
know what made the nonselective herbicide glyphosate (Roundup^TM)
a potent killer of so many different kinds of plants; weeds and crop plants as
well. It seemed reasonable that if you could alter crop plants so that they
were resistant to glyphosate, then spraying an herbicide like glyphosate “over
the top” of a mixture of emerging combination of resistant crops and weeds
would kill the weeds but not the crop.

Through combined research starting
early in the 1970’s glyphosate’s genetic “mode of action’, destruction of an
enzyme vital to all plants, was specifically identified by research performed
at Monsanto and by German researchers.

In 1983 researchers at Calgene and
Monsanto identified the gene, and Monsanto modified the gene, so that the
enzyme it produced was no longer sensitive to glyphosate. The A. tumefaciens
plasmid vector was used to
introduce the modified gene into crop plants. The new tomatoes, petunias and
other modified plants were resistant to damage from glyphosate which was
reported in the research in1985.

The USDA requires field testing of
genetically modified plants for several seasons before its release and that
testing includes potential changes in food safety, nutrient levels, development
of potentially toxic substances and safety to the environment. In 1996, the
first glyphosate-resistant soybean, cotton, canola, and corn seeds were made
available to farmers.

During the1980s and 1990s other
ways were developed to introduce beneficial genes into plants. One is called a
“gene gun,” which literally shoots DNA-covered particles attached to
metal “bullets” through plant cell walls and membranes to the cell nucleus.
Inside the nucleus the foreign DNA combines with the plant’s own DNA and
transforms the plant. Other techniques involve electrical or chemical
treatments assisting DNA molecules to pass through plant cell walls and
membranes barriers and combine with a plant’s genetic information, a high-tech
form of plant breeding. Wouldn’t Mendel be impressed?

Previously published in Southwest Trees and Turf